Bottom Line:
Lu et al.Therefore, it is possible that to silence the over expressed SPIK and thus to reinstate the activity of indispensable cellular proteases can result in the restoration of the susceptibility of HepG2 cells for HBV infection.The establishing a stable cell line for study of the early steps of HBV life cycle by silencing of SPIK is discussed.

ABSTRACTHepatitis B virus (HBV) is a human pathogen, causing the serious liver disease. Despite considerable advances in the understanding of the natural history of HBV disease, most of the early steps in the virus life cycle remain unclear. Virus attachment to permissive cells, fusion and penetration through cell membranes and subsequent genome release, are largely a mystery. Current knowledge on the early steps of HBV life cycle has mostly come from molecular cloning, expression of individual genes and studies of the infection of duck hepatitis B virus (DHBV) with duck primary duck hepatocytes. However, considering of the difference of the surface protein of HBV and DHBV both in the composition and sequence, the degree to which information from DHBV applies to human HBV attachment and entry may be limited. A major obstacle to the study HBV infection is the lack of a reliable and sensitive in vitro infection system. We have found that the digestion of HBV and woodchuck hepatitis virus (WHBV) by protease V8 led to the infection of HepG2 cell, a cell line generally is refractory for their infection [Lu et al. J Virol. 1996. 70. 2277-2285 . Lu et al. Virus Research. 2001. 73(1): 27-4].. Further studies showed that a serine protease inhibitor Kazal (SPIK) was over expressed in the HepG2 cells. Therefore, it is possible that to silence the over expressed SPIK and thus to reinstate the activity of indispensable cellular proteases can result in the restoration of the susceptibility of HepG2 cells for HBV infection. The establishing a stable cell line for study of the early steps of HBV life cycle by silencing of SPIK is discussed.

Figure 2: RT-PCR detects SPIK RNA. 1μg RNA from HepG2 cells and human liver cells were reversely transcripted and amplified by PCR using specific primer for SPIK. PCR bands were resolved in 1 % agarose gel and stain with ethidium Bromide. A: The names and location of primers. B: RT-PCR results. Lanes 1 to 4 were amplified with primers PI1/PI2; 5 to 8 were amplified with primers PI3/PI4. The samples from human liver cells were loaded in the column 1,2,5,6, (duplicated). The samples from HepG2 were loaded in the lane 3,4,7,8. Lane 9 was negative control without RTase. The specific SPIK bands are indicated by molecular weight. The lower bands in B are dimmer of primers. C: RT-PCR detection of TH gene of the human liver cells and HepG2 cells.

Mentions:
As shown in figure 2, two specific primer sets, PI1/ PI2 and PI3/PI4, were used to amplify SPIK mRNA by RT-PCR. PI1/PI2 directly amplifies the SPIK gene from the 11th bp to233th bp and PI3/PI4 directly amplifies the entire SPIK gene (Figure 2A). The RT-PCR band generated from primers PI3/PI4 was at the predicted size and slightly larger than that one generated from primers PI1/PI2 (324 base pairs versus 224 base pairs (Figure 2B). This suggests that the reactions of RT-PCR were specific. In addition, these bands were not generated from SPIK DNA (gene sequences), because the control that lacked the enzyme reverse transcriptase in the reaction mixture did not show any band (Figure 2B, lane 9). Compared to human liver cells, the expression of SPIK gene in the HepG2 cells was overwhelming. Using either of the primer sets, SPIK specific bands can be easily detected in the HepG2 cells, but hardly in the human liver cells (Figure 2B). This difference was not a result of the variation of loading, because the expression of the thimet oligopeptidase gene in the same condition did not show any difference (Figure 2C).

Figure 2: RT-PCR detects SPIK RNA. 1μg RNA from HepG2 cells and human liver cells were reversely transcripted and amplified by PCR using specific primer for SPIK. PCR bands were resolved in 1 % agarose gel and stain with ethidium Bromide. A: The names and location of primers. B: RT-PCR results. Lanes 1 to 4 were amplified with primers PI1/PI2; 5 to 8 were amplified with primers PI3/PI4. The samples from human liver cells were loaded in the column 1,2,5,6, (duplicated). The samples from HepG2 were loaded in the lane 3,4,7,8. Lane 9 was negative control without RTase. The specific SPIK bands are indicated by molecular weight. The lower bands in B are dimmer of primers. C: RT-PCR detection of TH gene of the human liver cells and HepG2 cells.

Mentions:
As shown in figure 2, two specific primer sets, PI1/ PI2 and PI3/PI4, were used to amplify SPIK mRNA by RT-PCR. PI1/PI2 directly amplifies the SPIK gene from the 11th bp to233th bp and PI3/PI4 directly amplifies the entire SPIK gene (Figure 2A). The RT-PCR band generated from primers PI3/PI4 was at the predicted size and slightly larger than that one generated from primers PI1/PI2 (324 base pairs versus 224 base pairs (Figure 2B). This suggests that the reactions of RT-PCR were specific. In addition, these bands were not generated from SPIK DNA (gene sequences), because the control that lacked the enzyme reverse transcriptase in the reaction mixture did not show any band (Figure 2B, lane 9). Compared to human liver cells, the expression of SPIK gene in the HepG2 cells was overwhelming. Using either of the primer sets, SPIK specific bands can be easily detected in the HepG2 cells, but hardly in the human liver cells (Figure 2B). This difference was not a result of the variation of loading, because the expression of the thimet oligopeptidase gene in the same condition did not show any difference (Figure 2C).

Bottom Line:
Lu et al.Therefore, it is possible that to silence the over expressed SPIK and thus to reinstate the activity of indispensable cellular proteases can result in the restoration of the susceptibility of HepG2 cells for HBV infection.The establishing a stable cell line for study of the early steps of HBV life cycle by silencing of SPIK is discussed.

ABSTRACTHepatitis B virus (HBV) is a human pathogen, causing the serious liver disease. Despite considerable advances in the understanding of the natural history of HBV disease, most of the early steps in the virus life cycle remain unclear. Virus attachment to permissive cells, fusion and penetration through cell membranes and subsequent genome release, are largely a mystery. Current knowledge on the early steps of HBV life cycle has mostly come from molecular cloning, expression of individual genes and studies of the infection of duck hepatitis B virus (DHBV) with duck primary duck hepatocytes. However, considering of the difference of the surface protein of HBV and DHBV both in the composition and sequence, the degree to which information from DHBV applies to human HBV attachment and entry may be limited. A major obstacle to the study HBV infection is the lack of a reliable and sensitive in vitro infection system. We have found that the digestion of HBV and woodchuck hepatitis virus (WHBV) by protease V8 led to the infection of HepG2 cell, a cell line generally is refractory for their infection [Lu et al. J Virol. 1996. 70. 2277-2285 . Lu et al. Virus Research. 2001. 73(1): 27-4].. Further studies showed that a serine protease inhibitor Kazal (SPIK) was over expressed in the HepG2 cells. Therefore, it is possible that to silence the over expressed SPIK and thus to reinstate the activity of indispensable cellular proteases can result in the restoration of the susceptibility of HepG2 cells for HBV infection. The establishing a stable cell line for study of the early steps of HBV life cycle by silencing of SPIK is discussed.